Multiple molecular binding sites

Abstract

The present disclosure relates to a support comprising a substrate and a plurality of sample spots on a surface of the support, wherein the sample spots are spatially separated from one another by the substrate, and a single biomolecule is immobilized on at least two of the separated sample spots.

Description

[Technical Field]

[0001] explanation The present disclosure relates to a support comprising a substrate and a plurality of sample spots on a surface of the support, wherein the sample spots are spatially separated from one another and a single biomolecule is immobilized on at least two of the separated sample spots. [Background technology]

[0002] background Sequencing the human genome or the genomes of other organisms, and determining and comparing individual sequence variations, requires the provision of sequencing methods that are firstly rapid and secondly can be employed routinely and cost-effectively.

[0003] The high demand for cost-effective sequencing has driven the development of high-throughput sequencing technologies that parallelize the sequencing process to generate multiple sequences simultaneously. Examples of these sequencing technologies include massively parallel signature sequencing (Lynx Therapeutics), polony sequencing (Life Technologies), 454 pyrosequencing (Roche Diagnostics), Illumina sequencing (Solexa Inc.), sequencing by ligation (Life Technologies), ion torrent semiconductor sequencing (Life Technologies), and DNA nanoball sequencing (Complete Genomics). These technologies enable rapid analysis of common sequences in a nucleic acid population. However, mutations present in a small number of sequences in the analyzed nucleic acid population, such as in a small number of cellular genomes, are often hidden by the large number of other sequences present in the population and therefore go undetected.

[0004] To address these problems, different types of single molecule analysis have been developed.Typically, for DNA or RNA sequencing, nucleic acid polymerase and / or nuclease and fluorescently labeled nucleic acid and / or nucleotide building blocks are used to individually determine the sequence of single nucleic acid molecules based on the time-dependent change in fluorescence when nucleotide building blocks are incorporated into or cleaved from nucleic acid molecules.Single molecule sequencing process and the device suitable for carrying out such process are described in, for example, co-owned patent applications WO 2002 / 097406, WO 2003 / 052137, WO 2006 / 013110, WO 2013 / 131888, WO 2015 / 104245, WO 2017 / 001407 and WO 2018 / 104301.

[0005] In some of these processes, nuclease molecules and / or nucleic acid synthesizing enzyme molecules, or complexes of such enzyme molecules with nucleic acid molecules, are provided in immobilized form on a solid support.

[0006] In addition to DNA and / or RNA sequencing, there are several additional applications where there is a high demand for single molecule analysis with high yield.

[0007] US 7,745,116 B2 (the contents of which are incorporated herein by reference) discloses compositions and methods for single molecule sequencing by detecting the incorporation of labeled nucleoside triphosphates into the growing end of a primer molecule. In particular, a polymerase-nucleic acid complex is provided that includes at least one anchor, for example, two anchors. The anchor can be selected from an amino acid, an epitope, a modified amino acid, a histidine tag, etc. The anchor can be used to attach a topological tether to the polymerase or to attach the polymerase to a support. There is no disclosure of attaching a single polymerase molecule to multiple discrete spots on a substrate.

[0008] It was an object of the present disclosure to provide supports containing immobilized biomolecules, including biomolecular complexes of precisely defined and / or advantageous configurations, suitable for the analysis of single molecule events, e.g., for the sequence analysis of single nucleic acid molecules. Summary of the Invention

[0009] Summary of the Invention In a first aspect, the present disclosure relates to a support comprising a substrate and a plurality of sample spots on a surface of the support, wherein the sample spots form a continuous region spatially separated from one another, and a single biomolecule is immobilized on at least two of the separated sample spots. In certain embodiments, the substrate is an optically transparent substrate.

[0010] A further aspect of the present disclosure relates to the use of the support described above for analyzing events, i.e., one or several events, at at least two sample spots, wherein the events are associated with the emission of electromagnetic radiation. In certain embodiments, the events are single molecule events. In some embodiments, analyzing events at at least two sample spots also includes analyzing events, i.e., one or several events between at least two sample spots.

[0011] Further aspects are: (i) providing a support having single biomolecules immobilized on at least two separate sample spots of the support; and (ii) analyzing events associated with said biomolecules, in particular single molecule events associated with said single biomolecules, by detecting electromagnetic radiation from said sample spots; The present invention relates to a method for analyzing an event, for example, a single molecule event, comprising:

[0012] In one embodiment, analyzing an event associated with a biomolecule by detecting electromagnetic radiation from at least two sample spots also includes detecting electromagnetic radiation from an area on the support between the at least two sample spots. In certain embodiments, a single molecule event comprises the sequencing of a single nucleic acid molecule.

[0013] Further aspects are: (i) a support on which a single biomolecule is immobilized on at least two separate sample spots of the support; (ii) means for irradiating at least two separate sample spots on the support; and (iii) means for analyzing events on said at least two sample spots by detecting electromagnetic radiation from said sample spots; The present invention relates to an apparatus adapted for analyzing events, for example single molecule events, including:

[0014] In one embodiment, the means for analyzing events on at least two sample spots associated with biomolecules by detecting electromagnetic radiation from at least two sample spots also includes means for detecting electromagnetic radiation from an area on the support between the at least two sample spots. In certain embodiments, the device is adapted for the sequence analysis of a single nucleic acid molecule.

[0015] Statement items 1. A support comprising a substrate and a plurality of sample spots on the support surface, wherein the sample spots are spatially separated from one another and biomolecules are immobilized on at least two separated sample spots.

[0016] 2. The support of item 1, in which biomolecules are immobilized on two, three or four separate sample spots, in particular on two separate sample spots.

[0017] 3. A support of item 1 or 2 that is at least substantially planar. 4. Support for item 1 or 2 being structured.

[0018] 5. The support of any one of the preceding items wherein the substrate is optically transparent. 6. The support of any one of the preceding items wherein the substrate comprises a material having a refractive index of at least 1.01.

[0019] 7. The support of any one of the preceding items, wherein the substrate comprises a non-conductive material. 8. The support of any one of the preceding items, wherein the substrate comprises a material selected from the group consisting of silica, quartz, and glass.

[0020] 9. The support of any one of the preceding items, wherein the substrate has a thickness of about 10 μm to about 5 mm, particularly about 20 μm to about 2 mm.

[0021] 10. The support of any one of the preceding items comprising at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1,000,000, or at least 10,000,000 sample spots.

[0022] 11. The support of any one of the preceding items, wherein at least one sample spot comprises at least one electrically conductive material, e.g., a metal, including a pure metal or a combination of metals, e.g., an alloy or mixture of multiple different metals.

[0023] 12. The support of item 11, wherein at least one metal is capable of forming a bond with sulfur, for example in the form of a thiol or disulfide.

[0024] 13. The support of any one of items 11 to 12, wherein at least the metal has a positive electrochemical potential.

[0025] 14. The support of any one of items 11 to 13, wherein at least one metal is selected from Au, Cu, Ni, Pt, Pd, Rh, Ir, Os, Ru, and any combination comprising at least two of said metals.

[0026] 15. The support of any one of items 1 to 10, wherein at least one sample spot comprises at least one metal oxide, such as TiO2, NiO, or ITO.

[0027] 16. The support of any one of the preceding items, wherein at least one sample spot has a diameter of about 1 nm to about 30 nm, in particular a diameter of about 2 nm to about 20 nm.

[0028] 17. The support of any one of the preceding items, wherein the sample spots are substantially uniformly distributed on the support surface.

[0029] 18. The support of item 17, wherein the distance between adjacent sample spots is about 1 nm to about 50 nm, for example, about 2 nm to about 20 nm.

[0030] 19. The support of any one of the preceding items, wherein the sample spots are distributed non-uniformly on the support surface.

[0031] 20. The support of item 19, wherein the sample spots are distributed on the support surface in groups of several spots, for example, about 2 to about 10 spots or about 2 to about 4 spots, and the distance between adjacent sample spots in different groups within a group is greater than the distance between adjacent sample spots in different groups.

[0032] 21. The support of item 20, wherein the distance between adjacent sample spots within a group is from about 1 nm to about 500 nm, for example, from about 2 nm to about 20 nm, or from about 20 nm to about 500 nm.

[0033] 22. The support of item 20 or 21, wherein the distance between adjacent sample spots in different groups is about 2 to about 2000 times greater, for example, about 5 to about 1000 times greater or about 10 to about 100 times greater, than the distance between adjacent sample spots in the same group.

[0034] 23. The support of any one of the preceding items, wherein the biomolecule is selected from the group consisting of polypeptides, nucleic acids, carbohydrates, and any combination thereof.

[0035] 24. A support according to any one of the preceding items, wherein the biological molecule is a nucleic acid polymerizing enzyme, particularly a DNA polymerase or an RNA polymerase, or a complex of a nucleic acid polymerizing molecule, particularly a DNA or RNA polymerizing complex comprising a nucleic acid polymerizing enzyme and a nucleic acid molecule.

[0036] 25. The support of item 24, wherein the biological molecule is a DNA polymerase having a DNA binding cleft, in particular a family A DNA polymerase including, but not limited to, Klenow, Taq, or T7 DNA polymerase, or any genetically modified version thereof, or a family B polymerase including, but not limited to, therminator, Phi29, RB-69, or T4 DNA polymerase, or any genetically modified version thereof.

[0037] 26. The support of any one of items 1 to 23, wherein the biological molecule is a gene editing enzyme, in particular a Cas nuclease such as Cas9 nuclease or any genetically modified version thereof, or a gene editing complex comprising a gene editing enzyme and a nucleic acid molecule, e.g., a guide RNA and / or a target nucleic acid.

[0038] 27. The support of any one of items 1 to 23, wherein the biological molecule is a nucleic acid molecule, such as a single-stranded or double-stranded nucleic acid molecule, in particular a DNA molecule or an RNA molecule. 28. The support of item 27, wherein the biomolecule is a single-stranded DNA or RNA molecule.

[0039] 29. The support of item 27 or 28, wherein the nucleic acid molecule has a length of at least about 10 nucleotides, at least about 100 nucleotides, at least about 1,000 nucleotides, for example, about 1,000 to about 100,000, particularly about 10,000 to about 50,000 nucleotides.

[0040] 30. The support of any one of the preceding items, wherein the biomolecule comprises at least two anchors attached to at least two separate sample spots.

[0041] 31. The support of item 30, wherein the anchor comprises a reaction product between a sulfur-containing group or a phosphorus-containing group and a metal or metal oxide surface on the sample spot, a reaction product between a reactive silane group and a metal oxide surface on the sample spot, or a reaction product between a poly(histidine) tag and a Ni or NiO surface on the sample spot.

[0042] 32. The support of item 30, wherein the anchor comprises an attached amino acid, or an attached tag, such as biotin, a hapten, a poly(histidine) tag, or a carbohydrate group, bound to a complementary portion of a coating attached to the sample spot surface, such as streptavidin, an antibody, or a lectin.

[0043] 33. The support of item 32, wherein at least one attached amino acid is selected from cysteine, modified phenylalanine, histidine, and glutamine.

[0044] 34. The support of item 30, wherein the anchor comprises the reaction product of two bio-orthogonal groups, for example, an azide group or an alkyne group. 35. The support of item 34, wherein the anchor comprises a triazole group.

[0045] 36. Use of the support of any one of items 1 to 35 for analyzing events occurring on at least two separate sample spots, said events being related to the emission of electromagnetic radiation.

[0046] 37. Use of item 36 for analyzing one or several events occurring on at least two separate sample spots and / or between at least two sample spots.

[0047] 38. Use of the support of any one of items 1 to 35 for analyzing single molecule events occurring on at least two separate sample spots, wherein said single molecule events are associated with the emission of electromagnetic radiation.

[0048] 39. Use of item 38 for analyzing one or several events occurring on at least two separate sample spots and / or between at least two sample spots.

[0049] 40. Use of the support of any one of items 1 to 35 for separately analyzing multiple events, in particular single molecule events each occurring on at least two separate sample spots, wherein said events are associated with the emission of electromagnetic radiation.

[0050] 41. Use of item 40 for analyzing multiple events occurring on at least two separate sample spots and / or between at least two sample spots, respectively.

[0051] 42. Use of items 40 or 41 in which multiple events, particularly single molecule events, are analyzed in parallel. 43. The use of any one of items 36 to 42, wherein the single molecule event comprises nucleic acid sequencing.

[0052] 44. Nucleic acid sequencing is - providing (i) a single nucleic acid molecule, (ii) molecules of a nucleic acid synthesizing enzyme and / or molecules of a nuclease immobilized on at least two separate sample spots, and (iii) fluorescently labeled nucleotide building blocks in free form and / or incorporated into the nucleic acid molecule; - performing an enzymatic reaction in which nucleotide building blocks are incorporated into and / or cleaved from said single nucleic acid molecule; and - individually determining the base sequence of nucleic acid molecules based on time-dependent fluorescence changes that occur when nucleotide building blocks are incorporated into and / or cleaved from said single nucleic acid molecule. Use of item 43, including:

[0053] 45. Use of item 44, wherein nucleic acid synthesis enzyme molecules and / or nucleic acid decomposition enzyme molecules are immobilized on the sample spots. 46. ​​Use of item 44, wherein a single nucleic acid molecule is immobilized on the sample spot.

[0054] 47. (iii) providing a support according to any one of items 1 to 35, wherein a single biomolecule is immobilized on at least two separate sample spots of the support; and (iv) analyzing events associated with said biomolecules, in particular single molecule events associated with said single biomolecules, by detecting electromagnetic radiation from said sample spots. A method for analyzing an event, for example a single molecule event, comprising:

[0055] 48. The method of item 47, wherein analyzing an event associated with a biomolecule by detecting electromagnetic radiation from at least two sample spots comprises detecting electromagnetic radiation from an area on the support between the at least two sample spots. 49. The method of item 47 or 48, wherein the single molecule event comprises sequencing of a single nucleic acid molecule.

[0056] 50. (iv) The support of any one of items 1 to 35, wherein a single biomolecule is immobilized on at least two separate sample spots of the support; (v) means for irradiating at least two separate sample spots on the support; and (vi) means for analyzing events on said at least two sample spots by detecting electromagnetic radiation from said sample spots; 1. An apparatus for analyzing an event, for example a single molecule event, comprising:

[0057] 51. The device of item 50, wherein the means for analyzing events on at least two sample spots associated with biomolecules by detecting electromagnetic radiation from at least two sample spots includes means for detecting electromagnetic radiation from an area on the support between the at least two sample spots. 52. The apparatus of item 50 or 51 adapted for sequence analysis of single nucleic acid molecules. [Brief explanation of the drawings]

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] Detailed Description The present disclosure relates to a support comprising a substrate and a plurality of sample spots spatially separated from one another on the support surface. In some embodiments, the support surface is formed by the substrate and the sample spots. In some embodiments, the substrate forms a continuous area in which the sample spots are distributed. Each sample spot on the support surface is surrounded by a substrate that is different from the sample spots, for example, in terms of material and / or surface. Typically, the substrate is adapted to inhibit and / or block attachment of biomolecules such as polypeptides, while the sample spots are adapted to allow attachment of desired biomolecules.

[0060] A single biomolecule is immobilized on at least two separate sample spots. The term "single biomolecule" encompasses a single molecular entity, such as a polypeptide or a complex consisting of multiple individual units, for example, an individual molecular entity in which the individual units together form a functional biological moiety. In some embodiments, a single biomolecule is immobilized on at least two, and up to 10, 100, 1,000, or 10,000, separate sample spots, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10, separate sample spots, particularly two separate sample spots. In some embodiments, a biomolecule, for example, a nucleic acid molecule of 1,000 nucleotides or more, may be immobilized on a large number of separate sample spots, for example, 100 or more sample spots.

[0061] In some embodiments, the support is a substantially planar support, i.e., it does not contain any protuberances or depressions of about 1000 nm or more, or about 100 nm or more. In further embodiments, the support is a structured support, e.g., a support having a surface area of ​​about 5×10 -24 liters ~ approx. 1 x 10 -15 The support includes a recess such as a well having a volume of 1 liter, or a pillar having a height of about 1 nm to about 500 nm. In principle, the support can have any design as long as a reaction space can be formed that allows the occurrence of a single molecule event on the at least two sample spots on which a single biomolecule is immobilized.

[0062] In certain embodiments, the substrate is an optically transparent material, i.e., a material that is substantially transparent to electromagnetic radiation, e.g., radiation in the visible range and / or radiation in the near-infrared range. In certain embodiments, the substrate comprises a material having an absolute refractive index of at least 1.01, e.g., an absolute refractive index of about 1.5 to about 3 in the visible range, or about 1.5 to about 4 in the near-infrared range. In further embodiments, the substrate is an optically opaque material, e.g., a metal or semi-metal such as silicon.

[0063] In certain embodiments, the substrate comprises a non-conductive material. Specific examples are glass, quartz, plastic, metal oxide-based materials, e.g., silicon dioxide-based materials such as glass, silica, or quartz, or composites comprising the foregoing. In further embodiments, the substrate comprises a conductive material, e.g., an optically transparent material such as indium tin oxide. Typically, the substrate has a thickness of about 10 μm to about 5 mm, particularly about 20 μm to about 2 mm.

[0064] In some embodiments, the substrate is coated with a layer of diamond-like carbon and / or amorphous carbon (also referred to as a "carbon film"), as described in co-pending application US 63 / 382,624 (the contents of which are incorporated herein by reference). The carbon film may be deposited on the substrate by physical vapor deposition, chemical vapor deposition (CVD), or atomic layer deposition (ALD) techniques. The carbon film may form a continuous layer on the support surface except in the area of ​​the sample spot. In some embodiments, the carbon film is fluorinated. Fluorine atoms may be introduced by known procedures, for example, by CVD during DLC ​​deposition or by exposure to a fluorine-containing plasma from a fluorocarbon-based gas (e.g., C4F8, CHF3, NF3, or SF6).

[0065] In one embodiment, the carbon film, including the fluorinated carbon film, has a thickness of about 0.3 nm to about 200 μm, particularly about 10 nm to about 100 μm, and more particularly about 1 μm to about 50 μm.

[0066] In certain embodiments, the substrate surface is coated with an organic passivating reagent, for example, a polyethylene glycol-containing reagent that inhibits and / or blocks the attachment of biomolecules such as proteins and / or nucleic acids.

[0067] The support surface comprises a plurality of sample spots spatially separated from one another by a substrate surface. The sample spots are adapted for attachment of biomolecules. In some embodiments, the support comprises a plurality of sample spots, e.g., at least 10, at least 100, at least 1,000, at least 10,000 sample spots, or at least 100,000 spots. In some embodiments, the support comprises at least 10 6 or 10 9 Pieces (up 10 6 or 10 9 ), or more sample spots.

[0068] In some embodiments, the sample spot comprises or consists of at least one conductive material, such as a single metal or a combination of metals, including alloys or mixtures of different metals. For example, metals that can be attached to sulfur-containing moieties (e.g., in the form of thiols or disulfides) or chelating moieties (e.g., polyhistidine tags) are suitable. In some embodiments, the metal has a positive electrochemical potential. Examples of suitable metals include, but are not limited to, Au, Cu, Ni, Pt, Pd, Rh, Ir, Os, Ru, and any combinations containing at least two of the foregoing metals.

[0069] In some embodiments, the sample spot comprises or consists of at least one metal oxide, including a single metal oxide or a combination of metal oxides. For example, metal oxides that can be attached to phosphorus-containing moieties (e.g., in the form of phosphonic acid or phosphonate esters) or chelating moieties (e.g., polyhistidine tags) are suitable. Examples of suitable metal oxides include TiO2 and NiO.

[0070] In a further embodiment, the sample spot comprises or consists of at least one non-conductive material.

[0071] Sample spots can be prepared by deposition of vaporized metal on a substrate covered with a grid mask, which can be fabricated by electron beam lithography or equivalent techniques. The size of the holes in the grid mask can correspond to the size of the spots on the substrate surface. Alternatively, spots on the substrate can be prepared by precision pipetting of particles onto the substrate, particularly on flat substrates, for example, by site-specific deposition of nanoparticles with a size of 2-10 nm.

[0072] The sample spot may have a size suitable for attachment of a single biomolecule as defined herein, and in certain embodiments, the sample spot has a diameter of about 1 nm to about 30 nm, particularly about 2 nm to about 20 nm.

[0073] In some embodiments, the sample spot is a discrete object on the substrate surface. In some embodiments, the sample spot has a lower surface near the substrate and an upper surface farther from the substrate, and the distance between the lower and upper surfaces defines the height of the sample spot. In certain embodiments, the height is about 50 pm to about 500 nm, particularly about 100 pm to about 20 nm, more particularly about 500 pm to about 10 nm, e.g., about 2 nm.

[0074] According to the present disclosure, the size and arrangement of the sample spots on the support surface are selected so that a single biomolecule is immobilized on at least two separate spots.

[0075] In some embodiments, the individual sample spots are substantially uniformly distributed on the support surface, and the distance between adjacent sample spots is about 1 nm to about 500 nm, e.g., about 2 nm to about 20 nm, or about 20 nm to about 500 nm, depending on the size of the immobilized biomolecule.

[0076] In some embodiments, the sample spots are distributed non-uniformly on the support surface. In these embodiments, the sample spots may be distributed on the support surface in groups of several spots, for example, about 2 to about 10 spots or about 2 to about 4 spots, where the distance between adjacent sample spots in different groups is greater than the distance between adjacent sample spots in different groups. In some embodiments, the distance between adjacent sample spots in a group is about 1 nm to about 50 nm, for example, about 2 nm to about 20 nm. The groups of sample spots may be distributed in a uniform or non-uniform pattern on the support surface.

[0077] The sample spots within a group may be separated from sample spots of adjacent groups by a distance that precludes immobilization of biomolecules on sample spots belonging to different groups. In some embodiments, the distance between adjacent sample spots in different groups is about 2 to about 2000 times greater, e.g., about 5 to about 1000 times greater, or about 10 to about 100 times greater, than the distance between adjacent sample spots in the same group.

[0078] The sample spots on the support are adapted for attachment of biomolecules, such that a single biomolecule is immobilized on at least two separate sample spots, for example, by covalent or non-covalent bonding. The biomolecule can be selected from polypeptides, nucleic acids, carbohydrates, and any combination thereof, for example, glycosylated polypeptides or ribonucleoproteins. In some embodiments, the biomolecule is a complex consisting of several individual units, for example, several polypeptide units, or several polypeptide and nucleic acid units.

[0079] In certain embodiments, the biomolecule is a nucleic acid polymerizing enzyme, particularly a DNA polymerase or an RNA polymerase, or a complex of nucleic acid polymerizing molecules, particularly a DNA or RNA polymerizing complex comprising a nucleic acid polymerizing enzyme and a nucleic acid molecule.In certain embodiments, the biomolecule is a DNA polymerase with a DNA binding cleft, particularly a family A DNA polymerase, including but not limited to Klenow, Taq or T7 DNA polymerase, or any genetically modified version thereof, or a family B polymerase, including but not limited to Therminator, Phi29, RB-69 or T4 DNA polymerase, or any genetically modified version thereof.In this context, refer to the aforementioned US 7,745,116 B2.

[0080] In a further embodiment the biomolecule is a nucleolytic enzyme, in particular an exonuclease, or a complex of nucleolytic molecules, in particular a complex of DNA or RNA degrading molecules comprising a nucleolytic enzyme and a nucleic acid molecule.

[0081] In another further embodiment, the biological molecule is a gene editing enzyme, in particular a Cas nuclease such as a Cas3, Cas9, Cas10, or Cas12 nuclease, or any genetically modified version thereof, e.g., a Cas nickase, or a gene editing complex comprising a gene editing enzyme and a nucleic acid molecule, e.g., a guide RNA and / or a target nucleic acid.

[0082] In another further embodiment, the biomolecule is a nucleic acid molecule, particularly a nucleic acid molecule as described in more detail below.

[0083] In some embodiments, the biomolecule comprises at least two anchors attached to at least two separate sample spots, e.g., two, three, or four anchors attached to at least two, e.g., two, three, or four separate sample spots.

[0084] In some embodiments, biomolecules are directly attached to sample spots, for example, by providing the biomolecule with an anchor suitable for direct covalent or non-covalent bonding to the sample spot surface, e.g., the inorganic surface of the sample spot. In these embodiments, the anchor may comprise a reaction product of a sulfur- or phosphorus-containing group, such as a thiol group -SH, a substituted thiol group -SR (where R is an organic residue, e.g., a C1-C4 alkyl group), or a disulfide group -SS-, with a metal or metal oxide surface on the sample spot. Alternatively, the anchor may comprise a reaction product of a silane group with a metal oxide, e.g., a silica surface, or a reaction product of a poly(histidine) tag with a Ni or NiO surface.

[0085] In some embodiments, the biomolecule is indirectly attached to the sample spot, for example, by non-covalent binding to a coating, e.g., an organic coating, on the sample spot surface. In these embodiments, the biomolecule may be provided with an anchor comprising an attached amino acid, such as cysteine, modified phenylalanine, histidine, or glutamine, or an attached tag, e.g., biotin, a hapten, a poly(histidine) tag, or a carbohydrate group, that can form a linkage with a complementary portion of the coating attached to the sample spot surface, e.g., streptavidin, an antibody, a lectin, etc.

[0086] In some embodiments, biomolecules can be provided with bioorthogonal groups, i.e., groups not present in biomolecules, such as azide or alkyne groups, e.g., terminal or strained alkyne groups such as cyclooctyne, that can form covalent bonds with complementary bioorthogonal groups attached to the sample spot surface. In these embodiments, the anchor can comprise the reaction product of a coupling reaction between two bioorthogonal reactive groups. In some embodiments, the coupling reaction is a click reaction, e.g., a reaction between an azide group and an alkyne group. In some embodiments, the anchor comprises a triazole group.

[0087] The supports of the present disclosure are suitable for analyzing events occurring on sample spots, wherein the events are associated with the emission of electromagnetic radiation from the sample spots. In certain embodiments, the events include biomolecular reactions associated with the emission of characteristic electromagnetic radiation. In certain embodiments, the events are single-molecule events. In some embodiments, analyzing the events involves directing electromagnetic radiation from a primary illumination source, e.g., a laser, onto the support to cause excitation of luminescent (e.g., fluorescent) groups associated with the events. Hereinafter, the terms "luminescent" and "fluorescent" are used interchangeably.

[0088] In certain embodiments, the support of the present disclosure is suitable for analyzing events, such as single molecule events occurring on at least one sample spot, particularly for separately analyzing multiple single molecule events each occurring on at least one sample spot, and more particularly for separately analyzing multiple single molecule events that are analyzed in parallel. In certain embodiments, the single molecule events include nucleic acid sequencing.

[0089] Further aspects are: (i) providing a support having single biomolecules immobilized on at least two separate sample spots on the support; and (ii) analyzing events associated with the single biomolecule by detecting electromagnetic radiation from the sample spot; The present invention relates to a method for analyzing an event, including:

[0090] In certain embodiments, the event is a single molecule event and the biomolecule is a single biomolecule. In certain embodiments, a single molecule event comprises the sequencing of a single nucleic acid molecule.

[0091] Further aspects are: (i) a support on which single biomolecules are immobilized on at least two separate sample spots of the support; (ii) means for irradiating at least two separate sample spots on the support; and (iii) means for analyzing events on said at least two sample spots by detecting electromagnetic radiation from said sample spots; The present invention relates to an apparatus adapted to analyze an event, comprising:

[0092] In certain embodiments, the event is a single molecule event and the biomolecule is a single biomolecule. In certain embodiments, the device is adapted for the sequence analysis of a single nucleic acid molecule.

[0093] Methods and apparatus for analyzing single molecule events are disclosed, for example, in WO 2002 / 097406, WO 2003 / 052137, WO 2006 / 013110, WO 2013 / 131888, WO 2015 / 104245, WO 2017 / 001407, and WO 2018 / 104301, the contents of which are incorporated herein by reference.

[0094] For the analysis of single molecule events, biomolecules are placed in sample spots on the support, where they are contacted with a sample liquid containing free reaction partners, thereby defining one or more reaction spaces. In particular, at least 100, at least 1000, or at least 10,000, and up to 10 6 More than one molecule can be analyzed on a single support, for example, a single planar support.

[0095] The nucleic acid molecules to be sequenced can be selected from DNA molecules, such as genomic DNA fragments, cDNA molecules, plasmids, or RNA molecules, such as mRNA molecules. The nucleic acid molecules can also be derived from genome or expression libraries generated from cells or organisms, e.g., eukaryotic or prokaryotic cells or organisms. This allows for the generation of multiple different nucleic acid template molecules, e.g., at least 10, 100, 1,000, or 10,000, and up to 100,000, 10 6 pieces or 10 7 This allows for the parallel sequencing of two or more different nucleic acid molecules.

[0096] The nucleic acid molecule to be sequenced can be linear or circular, for example, a covalently bonded circular single-stranded nucleic acid molecule.To obtain a circular nucleic acid template, the linear nucleic acid molecule can be subjected to a circularization procedure and optionally a strand separation procedure during sample preparation.Circularization can be carried out by ligation according to known protocols, for example, using DNA or RNA ligase.In some embodiments, adapter and / or identifier molecule, i.e., a nucleic acid molecule of known sequence, can be attached to the nucleic acid molecule.

[0097] Sequencing can involve nucleic acid extension and / or nucleic acid degradation. The sequencing process involves one or more sequencing cycles.

[0098] The nucleic acid polymerase molecule can extend a primer annealed to a nucleic acid template molecule. The primer extension can be achieved by stepwise incorporation of individual nucleotide building blocks into the 3'-end of the growing nucleic acid chain, thereby generating a nucleic acid molecule complementary to the sequence of the circular nucleic acid template. The nucleic acid polymerase is selected from polymerases capable of template-specific nucleic acid polymerization, preferably DNA polymerases and RNA polymerases, such as natural or modified polymerases including thermostable DNA polymerases, or reverse transcriptases.

[0099] Specific examples of suitable DNA polymerases include Taq polymerase, exonuclease-deficient Taq polymerase, E. coli DNA polymerase I, Klenow fragment, reverse transcriptase, Φ29-related polymerases, including wild-type Φ29 polymerase, and derivatives of such polymerases, such as exonuclease-deficient forms, T7 DNA polymerase, T5 DNA polymerase, RB69 polymerase, and the like.

[0100] Nuclease molecules can stepwise cleave individual nucleotide building blocks from nucleic acid molecules.Preferably, exonucleases are used, more preferably single-stranded exonucleases that degrade in the 3'→5' or 5'→3' direction.Particularly preferred exonucleases are 3'→5' exonucleases such as E. coli exonuclease I and E. coli exonuclease III, and 5'→3' exonucleases such as T7 exonuclease, E. coli exonuclease II, and E. coli exonuclease VIII.In addition, the exonuclease activity of various polymerases, such as Klenow fragment, Taq polymerase, or T4 polymerase, can also be used.

[0101] The nucleic acid synthesizing enzyme molecule is contacted with a linear or circular nucleic acid template molecule, such as a single-stranded DNA or RNA molecule, and a primer molecule annealed to or capable of annealing to the nucleic acid template molecule. The primer molecule is preferably a single-stranded nucleic acid or nucleic acid analog molecule with a free 3'-end that can be extended by an enzymatic reaction catalyzed by the immobilized nucleic acid synthesizing enzyme molecule. The length of the primer molecule is selected so that it can effectively anneal to the template under reaction conditions. Typically, the length of the primer molecule is at least 8, at least 10, at least 12, or at least 15 nucleotides, and for example, up to 20, 25, 50, or 100 nucleotides or more. In some embodiments, the primer is resistant to digestion by the nuclease molecule, for example, by incorporating a nucleotide analog building block and / or a linkage between the nucleotide building blocks, making it stable against degradation. In other embodiments, the primer is susceptible to digestion by the nuclease molecule.

[0102] The sequence of the primer is selected so that it effectively anneals to the template molecule under the reaction conditions. For example, the primer can be a universal degenerated primer that can statistically anneal to unknown nucleic acid sequences. In other embodiments, the primer can anneal to a known sequence portion of the nucleic acid template molecule. In this embodiment, a known adapter and / or identifier sequence may be incorporated into the nucleic acid template molecule. The primer can be unlabeled or contain a fluorescent labeling group.

[0103] Additionally, the presence of a nucleotide building block bearing at least one fluorescent labeling group is required. Preferably, each different nucleotide building block (A, G, C, T / U) contains a different luminescent (e.g., fluorescent) labeling group.

[0104] The luminescent (e.g., fluorescent) labeling group may be selected from known fluorescent labeling groups used to label biopolymers, particularly nucleic acids, such as, for example, fluorescein dyes, rhodamine, oxazines, e.g., Evoblue or Gnothis Blue, phycoerythrin, Cy3, Cy5, IR dyes or derivatives thereof.

[0105] A nucleotide building block may have (i) a fluorescent labeling group that remains with the building block when the building block is incorporated into a nucleic acid molecule during primer extension catalyzed by a molecule of nucleic acid synthesizing enzyme, and / or (ii) a fluorescent labeling group that is cleaved from the building block when the building block is incorporated into a nucleic acid molecule during primer extension catalyzed by a molecule of nucleic acid synthesizing enzyme. The fluorescent labeling group that remains with the building block is preferably attached to the α-phosphate group, sugar and / or nucleobase group.

[0106] In certain embodiments, the fluorescent labeling group that remains with the building block is attached to the nucleobase via a linker, which may have a chain length of, for example, up to 15, preferably 10-12, carbon atoms, and may optionally contain a heteroatom, such as an N, O, or S atom. The fluorescent labeling group that is cleaved when the building block is incorporated into a nucleic acid molecule may be attached to the terminal phosphate group of a polyphosphate building block, including, but not limited to, a hexa-, penta-, tetra-, or triphosphate building block, such as the γ-phosphate group of a triphosphate building block. In some embodiments, a building block is selected that contains both (i) a fluorescent labeling group that remains after incorporation and (ii) a fluorescent labeling group that is cleaved during incorporation. In this case, fluorescent groups that can interact with each other by, for example, quenching and / or energy transfer, may be selected.

[0107] The nucleic acid molecule to be sequenced contains a fluorescent labeling group when the nucleic acid molecule is subjected to direct sequencing using a molecule of nuclease, whereas the nucleic acid molecule to be sequenced may not contain a fluorescent labeling group when the nucleic acid molecule is used as a template in a primer extension.

[0108] The sequencing procedure may include a step of generating a nucleic acid molecule with incorporated nucleotide building blocks in primer extension catalyzed by a molecule of nucleic acid synthesizing enzyme, and / or a second step of cleaving individual nucleotide building blocks from the generated nucleic acid molecule catalyzed by a molecule of nuclease degrading enzyme. Depending on the type of luminescent (e.g., fluorescent) label, nucleic acid sequencing may be performed during primer extension and / or degradation.

[0109] Sequencing during primer extension involves the use of nucleotide building blocks bearing fluorescent labeling groups that are cleaved from the building blocks when incorporated into a nucleic acid molecule. In this case, time-dependent changes in fluorescence resulting from the cleavage of the fluorescent labeling groups from the nucleotide building blocks can be measured. Sequencing during nucleic acid degradation involves the use of nucleotide building blocks bearing fluorescent labeling groups that remain with the building blocks when incorporated into a nucleic acid molecule. The progressive cleavage of individual nucleotide building blocks from the nucleic acid molecule causes time-dependent changes in fluorescence as the labeled nucleotide building blocks are released from the nucleic acid molecule. In some embodiments, sequencing can also be performed during extension and degradation, i.e., when using nucleotide building blocks that have both a fluorescent labeling group that remains with the building block and a fluorescent labeling group that is cleaved from the building block when the building block is incorporated into a nucleic acid molecule. In this embodiment, both fluorescent groups can be the same or different.

[0110] In some embodiments, the method comprises one or more cycles of nucleic acid synthesis and nucleic acid degradation to determine the base sequence of a nucleic acid molecule template.Nucleic acid synthesis comprises the extension of a primer annealed to a nucleic acid template molecule, which is catalyzed by a molecule of nucleic acid synthesizing enzyme, thereby generating a nucleic acid molecule complementary to the sequence of the nucleic acid template.In the next step, the generated nucleic acid molecule is degraded by a molecule of nuclease decomposition.

[0111] When a nucleotide building block is incorporated into an elongated nucleic acid molecule, a time-dependent change in fluorescence can occur, which can be detected as described above. Preferably, the incorporation of a nucleotide building block into an elongated nucleic acid molecule is associated with a detectable increase in fluorescence, preferably a transient increase in fluorescence. For example, a nucleotide building block can be used that has a fluorescent labeling group on the part of the molecule that is cleaved when the building block is incorporated into a primer, for example, on the γ-phosphate group.

[0112] When nucleotide building blocks are cleaved from a synthesized nucleic acid molecule, a time-dependent change in fluorescence can be determined by the interaction of the fluorescent labeling group incorporated into the nucleic acid chain with neighboring groups, such as chemical groups of the nucleic acid, in particular nucleic acid bases, such as G, or / and with neighboring fluorescent labeling groups, which, by quenching or / and energy transfer processes, result in a change in fluorescence, particularly fluorescence intensity, compared to the "isolated" form of the fluorescent labeling group. The removal of individual nucleotide building blocks by cleavage changes the overall fluorescence, for example, the fluorescence intensity of the immobilized nucleic acid chain, and this change is a function of the removal of individual nucleotide building blocks by cleavage, i.e., a function of time.

[0113] In some embodiments, the association of the labeled nucleotide with the biomolecular complex is detected by measuring the polarization of the emitted photons. The polarization of the excited-state photons is altered by the rotational motion of the luminescent nucleotide label and can be used to distinguish free moving contra-bound labeled nucleotides during the polymerization process.

[0114] The time-dependent changes in fluorescence during this extension and / or degradation can be recorded in parallel for multiple nucleic acid molecules and correlated with the base sequences of the individual nucleic acid strands. It is preferred to use fluorescent labeling groups that are at least partially quenched when incorporated into a nucleic acid strand, such that the fluorescence intensity increases after the nucleotide building block containing the labeling group or the adjacent building block that causes quenching is removed by cleavage.

[0115] During the incorporation and / or removal of individual nucleotide building blocks, changes in the fluorescence intensity of the nucleic acid strand and / or the incorporated or cleaved nucleotide building blocks can be measured due to quenching or energy transfer processes. This time-dependent change in fluorescence intensity depends on the base sequence of the studied nucleic acid strand and can therefore be correlated with the sequence.

[0116] By using a mixture of nucleotide building blocks labeled with all four different bases, e.g., A, G, C, and T, or combinations of two or three different bases, the complete sequence of a nucleic acid molecule can be determined. Where appropriate, a "sequence identifier," i.e., a labeled nucleic acid of known sequence, can also be attached to the nucleic acid strand being studied, e.g., by an enzymatic reaction using ligase and / or terminal transferase, so that at the start of sequencing, first a known fluorescence pattern is obtained, and only thereafter a fluorescence pattern corresponding to the unknown sequence being studied is obtained.

[0117] Detection involves irradiating the support with light, preferably by a laser or another suitable light source, to cause excitation of the fluorescent labeling group. In this regard, one or more laser beams, e.g., an expanded laser beam having a cross section of about 1 to 20 mm, and / or multiple laser beams, can be used. Detection preferably involves multipoint fluorescence excitation by laser, e.g., a dot matrix of laser dots generated via diffraction optics (see WO 2002 / 097406) or quantum well lasers.

[0118] The fluorescence emission of multiple nucleic acid strands can be detected in parallel, for example, using an electronic detector matrix, such as a CCD camera, a CMOS detector matrix, or a detector matrix including an avalanche photodiode matrix. Detection can be performed so that fluorescence excitation and detection are performed in parallel for some or all of the nucleic acid strands being studied. It is preferable to perform detection on the fluorescence emitted essentially orthogonally from the support surface, through the reaction space, or through the support body.

[0119] Detection can be performed, for example, by single molecule detection, for example, by fluorescence correlation spectroscopy, which can be performed using very small, preferably confocal, volume elements (e.g., 10 -21 ~10 -101) to excitation light from a laser or another suitable light source, which excites receptors present in the measurement volume, causing the latter to emit fluorescence, and the fluorescence emitted from the measurement volume is measured by a photodetector, and the time course of the measured emission is correlated with the concentration of the analyte, so that at suitable high dilutions, individual molecules in the measurement volume can be identified. Details of the procedure and the equipment used for detection can be found in the disclosure of EP 0 679 251, the contents of which are incorporated herein by reference. Confocal determination of single molecules is further described in Rigler and Mets (Soc. Photo-Opt. Instrum. Eng. 1921 (1993), 239 ff.) and Mets and Rigler (J. Fluoresc. 4 (1994) 259-264), the contents of which are incorporated herein by reference.

[0120] Alternatively or additionally, detection can also be performed by time-resolved decay measurements, called "time gating," as described, for example, by Rigler et al., "Picosecond Single Photon Fluorescence Spectroscopy of Nucleic Acids" in "Ultrafast Phenomena," D.H. Auston, Ed., Springer 1984 (the contents of which are incorporated herein by reference). Here, fluorescent molecules are excited within the measurement volume and then detected on a photodetector at a time interval of, for example, ≥ 100 ps. In this way, the background signal generated by the Raman effect can be kept sufficiently low, allowing single molecules to be detected in an essentially interference-free manner.

[0121] In certain embodiments of the present disclosure, the single biomolecule attached to the plurality of sample spots is a nucleic acid molecule, for example, a single-stranded or double-stranded nucleic acid molecule. In certain embodiments, the single biomolecule is a single DNA molecule or a single RNA molecule.

[0122] In certain embodiments, the nucleic acid molecule has a length of at least about 10 nucleotides, at least about 100 nucleotides, at least about 1,000 nucleotides, e.g., about 1,000 to about 100,000, particularly about 10,000 to about 50,000 nucleotides, e.g., deoxyribonucleotide building blocks, ribonucleotide building blocks, and / or nucleotide analog building blocks.

[0123] A typical length of a nucleic acid molecule, such as a DNA or RNA molecule, is the length of a human gene, which is, on average, about 15,000 nucleotides. One base has a length of about 0.3 nm. Nucleic acid molecules having shorter or longer lengths can also be used for certain applications.

[0124] In those embodiments, methods and devices are provided for the parallel read-out of the sequences of several different portions of a single DNA or RNA molecule.

[0125] The nucleic acid molecule, e.g., DNA or RNA molecule, comprises at least one anchor, particularly at least two or more anchors, for immobilizing it on a support having a surface containing a plurality of sample spots, e.g., metal spots, as described above. In certain embodiments, only one anchor is bound to one sample spot.

[0126] In some embodiments, the anchor is suitable for directly or indirectly attaching a nucleic acid molecule to a sample spot, as described above, and in those embodiments, the biomolecule may have a bioorthogonal group, as described above.

[0127] In certain embodiments, the anchor comprises a capture probe comprising a nucleic acid sequence complementary to a sequence portion of a single nucleic acid molecule immobilized on a sample spot. The capture probe forms a double-stranded hybrid with the single nucleic acid molecule, thereby immobilizing the single nucleic acid molecule on the sample spot to which the probe is attached. The length of the complementary sequence in the capture probe is sufficient to allow stable hybridization when analyzing single-molecule events.

[0128] In some embodiments, the length of the complementary sequence in the capture probe is at least about 10, at least about 12, at least about 14, and at least about 16 nucleotides (including nucleotide analogs). In some embodiments, the length of the complementary sequence in the capture probe is up to about 100, up to about 80, up to about 60, and up to about 40 nucleotides (including nucleotide analogs). At least two, for example, two, three, four, five, six, seven, eight, nine, ten, or more different capture probes can be used to immobilize a single nucleic acid molecule. Different capture probes can hybridize to different portions of a single nucleic acid molecule. The portions to which the capture probes can hybridize can be selected specifically or randomly.

[0129] In some embodiments, the fixed single nucleic acid molecule serves as a template strand for a nucleic acid polymerization process, in which a new nucleic acid strand complementary to the template strand is generated by primer extension. In these embodiments, one or more or all of the anchors have primer functionality, i.e., as described above, they are 3' starting points for nucleic acid polymerizing enzymes, such as DNA polymerase, RNA polymerase, or reverse transcriptase. For this purpose, the anchors may have a capture probe sequence with a free 3'-end accessible to the nucleic acid polymerizing enzyme. Polymerization involves the incorporation of individual nucleotide building blocks into the new nucleic acid strand. This incorporation event results in the emission of electromagnetic radiation, which can be detected as described above.

[0130] In embodiments in which the immobilized single nucleic acid molecule is the template strand for a nucleic acid polymerization process, the nucleic acid polymerizing enzyme is typically present in the reaction medium as a free molecule, i.e., not immobilized to a sample spot on a support, and the reaction medium further contains luminescent (e.g., fluorescent) nucleotide building blocks for incorporation into new nucleic acid strands generated by primer extension.

[0131] In certain embodiments, detection of emitted electromagnetic radiation occurs with approximately equal sensitivity over the entire surface of the support, i.e., the area of ​​the sample spots and the areas between the sample spots, and in these embodiments, the optical focus of detection of emitted electromagnetic radiation is directed not only at emitted electromagnetic radiation originating from the area of ​​each individual sample spot, but also at the areas between the sample spots.

[0132] The primary radiation source can be directed toward the support such that a substantially uniform or uniform primary radiation field is generated at the surface, thereby irradiating all molecules located at or near the surface. The intensity of the primary radiation field is typically measured in a direction perpendicular to the surface, e.g., r -2 where r is the distance to the surface in the orthogonal direction), so that the primary radiation field is greatest near the surface.

[0133] Immobilizing nucleic acid molecules, such as DNA or RNA molecules, at multiple sample spots on a support using several anchors allows the entire nucleic acid molecule to be positioned approximately horizontally relative to the support surface. Between the individual attachment positions, the nucleic acid molecule can bend to a limited extent in all three dimensions. This allows for efficient and sensitive detection of the electromagnetic radiation emitted by the incorporation of nucleotide building blocks along the entire length of the immobilized nucleic acid molecule.

[0134] The sequence of a single DNA or RNA molecule can be read in parallel for each portion defined by each anchor. The number of anchors used for a nucleic acid molecule correlates with the degree of parallelism that can be obtained for a molecule. The readout speed of an individual nucleic acid molecule, for example, a DNA or RNA molecule, is roughly proportional to the number of anchors. Therefore, using a large number of anchors, for example, one anchor per about 50 to about 1,000 nucleotides or more, can achieve a very high readout speed for sequencing DNA or RNA molecules.

[0135] The disclosed methods and apparatus are also suitable for the analysis of additional single molecule events, i.e., single biomolecules bound to selected spots for analysis, where high yield single molecule analysis is in high demand.

[0136] In certain embodiments, the present disclosure relates to single molecule analysis of receptor-ligand interactions, for example, involving binding of a receptor protein to a sample spot and then studying its interaction with its ligand, for example, within drug development.

[0137] In further embodiments, the present disclosure relates to single-molecule analysis of hybridization events, including the attachment of short, single-stranded nucleic acid molecules, e.g., DNA or RNA molecules, having lengths in the range of 3-300 nucleotides, followed by the addition of a sample containing a complementary nucleic acid molecule and observing any hybridization events. Applications can include, for example, viral RNA / DNA detection, bacterial DNA / RNA detection, and detection of short segments of DNA from cancer cells in the bloodstream.

[0138] The present disclosure will be further described in detail with reference to the following specific embodiments. Figure 1 shows an embodiment of the prior art: a support (1) contains a sample spot (2) to which a single biomolecule (3), e.g., a DNA polymerase molecule, is immobilized via anchors (4a, 4b), both of which are fixed to the same sample spot.

[0139] Figure 2 shows an embodiment of the present disclosure. A support (1) includes two sample spots (2a, 2b) on which single biomolecules (3), e.g., DNA polymerase molecules, are immobilized via anchors (4a, 4b). Each anchor is immobilized on a separate sample spot. The diameter of the sample spots is, for example, in the range of 1 to 30 nm. The distance between adjacent spots is, for example, in the range of 1 to 50 nm.

[0140] Figure 3 shows an embodiment of the present disclosure. The support (1) contains four sample spots (2a, 2b, 2c, and 2d), on which single biomolecules (3), such as nucleic acid polymerization complexes or gene editing complexes consisting of multiple individual units, are immobilized via anchors (4a, 4b, 4c, and 4d). Each anchor is fixed to a separate bond. The diameter of the sample spots ranges, for example, from 1 to 30 nm. The distance between adjacent spots ranges, for example, from 30 to 1,500 nm.

[0141] Figure 4 illustrates the preparation of single-stranded DNA or RNA molecules (10) from a sample (12). Suitable samples include, but are not limited to, biological samples such as blood, animal, e.g., human tissue, hair, and cultured cells. An anchor (14) containing a capture probe sequence (16) is attached to the DNA or RNA molecule. In some embodiments, the capture probe sequence specifically binds to a portion of the DNA or RNA molecule. Alternatively, the capture probe sequence binds stochastically and nonspecifically to the DNA or RNA molecule. This results in a DNA or RNA molecule (18) with the anchor attached thereto.

[0142] 5 shows an exemplary anchor 14 attached to a DNA or RNA molecule 20. The anchor 14 includes a capture probe 22 having the sequence 5'-TAC-3' complementary to a portion 5'-...GTA...3' of the DNA or RNA molecule 20, an accessible 3'-end, and an attachment group 24. The capture probe 22 is attached to the DNA or RNA molecule 20, and the attachment group 24 is attached to a sample spot.

[0143] 6 shows an exemplary support comprising a surface containing m×n sample spots separated from each other by a distance a or b and having a diameter d, where the parameters a, b, d, and m×n are as described herein above.

[0144] FIG. 7 shows an exemplary DNA or RNA molecule (18) immobilized on a plurality of sample spots (26a, 26b, 26c, 26d, 26e, 26f) and having an anchor attached thereto.

[0145] Figure 8 is a schematic depiction of the polymerization process for the DNA or RNA molecule (18) in Figure 7. Several polymerase molecules (28a, 28b, 28c, 28d, and 28e) simultaneously catalyze polymerization reactions at different portions of the DNA or RNA molecule (18), thereby producing complementary nucleic acid strands (30a, 30b, 30c, 30d, and 30e). Polymerase molecules can be added to the immobilized DNA or RNA molecule. The polymerase molecules freely diffuse into the medium surrounding the immobilized DNA or RNA molecule until they bind to the 3'-end of a capture probe. Nucleotides of all four base categories, A, T / U, G, and C, also freely diffuse into the solution surrounding the DNA or RNA molecule. Once the polymerase molecule attaches to the 3'-end of the capture probe, polymerization is initiated and continues until the polymerase reaches the next capture probe.

[0146] 9 shows further examples of DNA or RNA molecules (18) attached to sample spots (26a, 26b, 26c, 26d, 26e, 26f, 26g) via anchors containing attachment groups. In some applications, not all attachment groups may need to be bound to the sample spots (not shown).

[0147] Figure 10 shows several DNA or RNA molecules attached to a surface. This figure illustrates this situation for two molecules (32, 34). In some embodiments, the number of molecules attached to the surface in a single measurement is approximately 1,000, but could be 10,000, 100,000, 1,000,000, or even 100,000,000. In some applications, only a small number of molecules may be desired, and therefore fewer than 1,000, typically 1, 10, or 100 molecules, are attached to the surface simultaneously. Partial overlap between different molecules may reduce accuracy only in such overlapping regions. In non-overlapping regions, the simultaneous presence of many different molecules on the surface does not adversely affect accuracy.

Claims

1. A support comprising a substrate and a plurality of sample spots on the support surface, wherein the substrate forms a continuous area in which the sample spots are spatially separated from one another, and a single biomolecule is immobilized on at least two separate sample spots.

2. 10. The support of claim 1, comprising at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1,000,000, or at least 10,000,000 sample spots.

3. 10. The support of claim 1, wherein at least one sample spot has a diameter of about 1 nm to about 30 nm, in particular a diameter of about 2 nm to about 20 nm.

4. 10. The support according to claim 1, wherein the sample spots are substantially uniformly distributed on the support surface, the distance between adjacent sample spots being in particular from about 1 nm to about 500 nm, for example from about 2 nm to about 20 nm, or from about 20 nm to about 500 nm.

5. 4. The support according to claim 1, wherein the sample spots are distributed unevenly on the support surface, and the sample spots are particularly distributed on the support surface in groups of several spots, for example, about 2 to about 10 spots or about 2 to about 4 spots, and the distance between adjacent sample spots in different groups within a group is greater than the distance between adjacent sample spots in different groups.

6. The support of claim 5, wherein the distance between adjacent sample spots within a group is from about 1 nm to about 50 nm, for example, from about 2 nm to about 20 nm, and / or the distance between adjacent sample spots in different groups is from about 2 to about 2000 times greater, for example, from about 5 to about 1000 times or from about 10 to about 100 times greater, than the distance between adjacent sample spots in the same group.

7. the biomolecule is selected from the group consisting of a polypeptide, a nucleic acid, a carbohydrate, and any combination thereof; the biomolecule is in particular a nucleic acid polymerizing enzyme, in particular a DNA polymerase or an RNA polymerase, or a complex of nucleic acid polymerizing molecules, in particular a complex of DNA or RNA polymerizing molecules comprising a nucleic acid polymerizing enzyme and a nucleic acid molecule, or the biological molecule is in particular a gene editing enzyme, in particular a Cas nuclease such as Cas9 nuclease or any genetically modified version thereof, or a gene editing complex comprising a gene editing enzyme and a nucleic acid molecule, e.g. a guide RNA and / or a target nucleic acid; or The biological molecule is, in particular, a nucleic acid molecule, for example, a single-stranded or double-stranded single-stranded DNA or RNA molecule, and the nucleic acid molecule has a length of at least about 10 nucleotides, at least about 100 nucleotides, at least about 1,000 nucleotides, for example, about 1,000 to about 100,000 nucleotides, in particular, about 10,000 to about 50,000 nucleotides. A support according to any one of the preceding claims.

8. 10. A support according to any one of the preceding claims, wherein the biomolecule comprises at least two anchors attached to at least two separate sample spots.

9. 9. Use of a support according to any one of claims 1 to 8 for analyzing events, in particular single molecule events occurring on at least two separate sample spots, said events being associated with the emission of electromagnetic radiation.

10. 9. Use of a support according to any one of claims 1 to 8 for separately analysing a plurality of events, in particular single molecule events, each occurring on at least two separate sample spots, wherein said events are associated with the emission of electromagnetic radiation, and said plurality of events are analysed, in particular in parallel.

11. Single molecule events - providing (i) a single nucleic acid molecule, (ii) molecules of a nucleic acid synthesizing enzyme and / or molecules of a nuclease immobilized on at least two separate sample spots, and (iii) fluorescently labeled nucleotide building blocks in free form and / or incorporated into the nucleic acid molecule; - performing enzymatic reactions in which nucleotide building blocks are incorporated into and / or cleaved from said single nucleic acid molecule; and - individually determining the base sequence of nucleic acid molecules based on time-dependent fluorescence changes that occur when nucleotide building blocks are incorporated into and / or cleaved from said single nucleic acid molecule; The use according to claims 9 to 10, comprising nucleic acid sequencing comprising:

12. (v) providing a support according to any one of claims 1 to 8, wherein a single biomolecule is immobilized on at least two separate sample spots of the support; and (vi) analyzing events associated with said biomolecules, in particular single molecule events associated with said single biomolecules, by detecting electromagnetic radiation from said sample spots. A method for analyzing an event, for example a single molecule event, comprising:

13. 13. The method of claim 12, wherein the single molecule event comprises sequencing of a single nucleic acid molecule.

14. (vii) a support according to any one of claims 1 to 8, wherein a single biomolecule is immobilized on at least two separate sample spots on the support; (viii) means for irradiating at least two separate sample spots on the support; and (ix) means for analyzing events on said at least two sample spots by detecting electromagnetic radiation from said sample spots; 1. An apparatus for analyzing an event, for example a single molecule event, comprising:

15. 15. The device of claim 14 adapted for sequence analysis of a single nucleic acid molecule.

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